ProjectSummary Themicroenvironmentplaysauniqueroleinthedevelopmentofmelanoma,whicharisesintheskin.Theskin displays externally visible signs of aging, such as decreased elasticity and a loss of collagen integrity. Much workhasfocusedrecentlyonhowchangesincollagencanaffecttumormetastasis,bothbiomechanicallyand biochemically. Data from several groups indicate that the cross-talk between stromal fibroblasts and transformed melanocytes is important for invasion, melanoma growth, and even therapy resistance. Our proteomicsanalysesindicatethatfactorsusedtocrosslinkcollagen,suchasHAPLN1aresecretedbyyoung, but not aged dermal fibroblasts, and Wnt5A is secreted by aged, but not young fibroblasts. The interplay between these molecules may contribute to age-related changes in collagen integrity. We hypothesize that changes regulated by age-related alterations in the extracellular matrix (ECM) initiate or promote a pro- metastaticprogram,andimpacttherapyresistance. We will query the contribution of aged skin biochemical and architectural contributions in governing melanoma?smetastaticprogressionaswellasresistancetotargetedtherapy.Weproposeanalternatetheory of matrix stiffness that hypothesizes that increasing stiffness will have a non-linear effect on metastatic progression,andtherapyresistance.Wewillusepathophysiologicallyrelevantinvitro3Dstromalsystems(3D skin reconstructions), animal models of melanoma, a novel simultaneous multi-channel immunofluorescent analysis (SMIA) of formalin-fixed and paraffin embedded (FFPE) human tissue cohorts in combination with a customized new software written for the bulk analysis and acquisition of SMIA-generated images, single moleculeRNAimagingcoupledwithhigh-throughputsinglecellimagingandsequencing,allofwhichwillfeed back into an increasingly intricate mathematical modeling for improved understanding and better patient personalized (i.e., metastatic and efficacy of drug treatment) prediction capabilities. We expect our data will reveal a synergistic picture of the mechanochemical interactions between the aged microenvironment and singulartumorcellsthattheindividualapproachescouldnothavedeciphered. This team project involves experts in the biology of melanoma metastasis, Wnt signaling and aging (Weeraratna), single cell RNA systems biology (Raj), tumor-stromal interactions and digital imaging quantitativeanalyses(Cukierman)andcomputationalandmathematicalpredictivemodeling(Shenoy).